Effect of Wenxin granule on Bcl-2/Bax/Caspase apoptosis pathway gene expression in rats with myocardial infarction

Objective:To explore the cardioprotective mechanism of Wenxin Granules regulating the expression of apoptosis-related genes in cardiomyocytes.Methods:A rat model of myocardial infarction was established and randomly divided into model group,Wenxin granule low-dose group,Wenxin granule high-dose group,metoprolol group and sham operation group.the left ventricular end systole anterior wall thickness(LVAWs),end systole inner diameter(LVIDs),end systole posterior wall thickness(LVPWs),end-diastolic anterior wall thickness(LVAWd),end-diastolic inner diameter(LVIDd),end-diastolic posterior wall thickness(LVPWd)and left ventricular ejection fraction(LVEF)were detected by echocardiography in each group after 2 weeks of treatment.Hematoxylin eosin(HE)staining was used to observe the changes in the cardiac structure of rats in each group.Real-time PCR(Real-time PCR)was used to detect the relative expression of mammalian B-cell lymphoma-2(BCL-2),BCL-2 related X protein(BAX),Caspase-9(Caspase-9),and Caspase-3(Caspase-3)mRNA.TUNEL staining was used to detect changes in the apoptotic rate of rat cardiomyocytes in each group.Results:Compared with the sham operation group,the LVAWs,LVPWs,LVPWd and LVEF of the model group were significantly reduced(P<0.05,P<0.01),and LVIDs and LVIDd were significantly increased(P<0.05,P<0.01).Severe pathological ischemia injury of heart tissue.The relative expression of BCL-2 mRNA and the ratio of BCL-2/BAX in the model group were significantly reduced(P<0.01),while the relative expression of BAX,Caspase-9 and Caspase-3 mRNA was significantly increased(P<0.01).The apoptosis rate was significantly increased(P<0.01).In the low-dose and high-dose groups of Wenxin Granules and the Metoprolol group,LVAWs,LVPWs,LVPW d,and LVEF of rats in each administration group increased significantly(P<0.05,P<0.01),LVIDs,LVIDd was significantly reduced(P<0.05,P<0.01),the pathological damage of the heart tissue was improved,the expression of BCL-2 mRNA and the ratio of BCL-2/BAX were significantly increased(P<0.05,P<0.01),BAX,The expression of Caspase-9 and Caspase-3 mRNA was significantly reduced(P<0.05,P<0.01),and the apoptotic rate of myocardial cells was significantly reduced(P<0.01).Conclusion:Wenxin granule can play a cardioprotective role by regulating the gene expression of BCL-2/BAX/Caspase apoptosis pathway.

Differential expression of bcl-2 and bax genes during the E.coli-induced apoptosis of human U937 cells

To explore the role of bcl-2 and bax genes in the apoptosis of human U937 cells induced by E.coli, flow cytometry assay with annexinⅤ-FITC/PI double staining was used to determine the condition of apoptosis, and the expressions of mRNA of bcl-2 and bax genes were assayed with RT-PCR. It was found that the apoptosis of human U937 cells could be induced by E.coli at various concentration ratios between cells and bacteria for 30 min in a dose-dependent manner. The apoptotic rates at cell/bacteria ratios of 0, 1∶5, 1∶10, 1∶20, 1∶50 and 1∶100 were 3.16%±0.90%, 9.46%±0.84%, 17.90%±1.41%, 35.59%±3.76%, 38.35%±7.12% and 55.07%±5.82% respectively. Also, there was a tendency of alterations in the expression levels of bcl-2 and bax genes with an increased expression level of bax gene and a reduced expression level of bcl-2 gene. It is concluded that E.coli can induce apoptosis in human U937 cells with a down-regulated expression of Bcl-2 and an up-regulated expression of Bax, and this might be related to the induction of apoptosis of the infected cell.

EFFECT OF ELECTROACUPUNCTURE ON NOREPINEPHRINE LEVEL AND APOPTOSIS IN CEREBRAL CORTEX TISSUE IN RATS WITH CEREBRAL ISCHEMIA-REPERFUSION

Objective: To investigate the underlying neurobiological mechanism of the protective effect of electroacupuncture (EA) during cerebral ischemia-reperfusion (CI-R). Methods: In the first part of the study, 15 SD rats were evenly randomized into control group, CI-R-48h model group and CI-R-48h+EA group. The cortical apoptosis and expression of Bcl-2 and Bax proteins in each group were detected by flow cytometer (FCM). In the second part of the study, 75 SD rats were evenly randomized into control, CI-R-3min, CI-R-3min+EA, CI-R-48h and CI-R-48h+EA groups. Cortical norepinephrine (NE) concentration was detected by fluorescence spectrometer. CI-R model was established by occlusion of the bilateral common carotid arteries and reperfusion. EA (4～16 Hz, 1～3 V) was applied to “Shuigou”(水沟 GV 26) and “Chengjiang”(承浆 CV 24) for 30 min before CI and after reperfusion respectively. Results: In the first part of this study, results indicated that the number of the apoptotic neurons and the apoptosis rate of CI-R-48h group were significantly higher than those of control group; while comparison between CI-R-48h+EA and CI-R-48h groups showed that the number of the apoptotic neurons and the apoptosis rate of the former group were significantly lower than those of the later group (P<0.05). In comparison with control group, after CI-48h, Bax expression was up-regulated significantly and Bcl-2 down-regulated markedly (P<0.05). Comparison between CI-R-48h and CI-R-48h+EA group indicated that Bax expression of the later group was significantly lower than that of the former group, while Bcl-2 expression of CI-R-48h+EA group was significantly higher than that of CI-R-48h group (P<0.05), suggesting that EA could reverse CI induced reactions of these two indexes. In the second part of the study, in comparison with control group, NE concentration in cerebral cortex of CI-R-3min group increased significantly (P<0.05); while NE content of CI-R-3min+EA group was significantly lower than that of CI-R-3min group (P<0.05). No significant difference was found between CI-R-3min group and control group in cortical NE levels; and no significant changes were found about NE levels in CI-R-48h and CI-R-48h+EA groups, suggesting that EA could inhibit the increase of cortical NE level in the early stage of CI. Conclusion: Changes of NE concentration in the cerebral cortex during the earlier period of CI-R is possibly related to the incidence of cortical apoptosis. EA can reduce the increase of NE due to CI and thus may inhibit CI-induced cortical apoptosis.

Melatonin restores normal Bax and Bcl-2 protein expression in the subgranular zone of the dentate gyrus in pinealectomized rats

In this study, we sought to elucidate the effects of melatonin on learning and memory as well as apoptosis and expression of the Bax or Bcl-2 proteins in the subgranular zone of the dentate gyrus in pinealectomized rats. Using the Morris water maze and the olfactory memory tests, we found that the average escape latency in pinealectomized rats was clearly increased compared with sham-operated rats. Moreover, the average escape latency in the melatonin-treated and pinealectomized rats was longer than that in the sham-operated rats and shorter than that in the pinealectomized and untreated rats. Immunohistochemistry and terminal-deoxynucleoitidyl transferase mediated nick end labeling （TUNEL） showed that there were fewer Bax immunoreactive cells and TUNEL-positive （apoptotic） cells but more Bcl-2 immunoreactive cells in the melatonin-treated rats compared with the pinealectomized rats. The sham-operated rats showed numbers of these cells similar to the melatonin-treated rats. These experimental findings demonstrate that melatonin treatment may reduce abnormal apoptosis by promoting gene expression of Bax and suppressing gene expression of Bcl-2 in the subgranular zone of the dentate gyrus in pinealectomized rats. These effects appear to result in the inhibition of cellular apoptosis and the improvement of spatial learning and memory in pinealectomized rats.

(-)-Epigallocatechin-3-gallate protects spiral ganglion neurons against amikacin-induced apoptosis

Morphology of spiral ganglion neurons （SGNs） in Sprague-Dawley rats before and after amikacin treatment was observed by transmission electron microscopy. Amikacin induced cochlear SGN apoptosis. Immunohistochemical staining and RT-PCR revealed a decrease in Bcl-2 protein ex-pression, and an increase in Bax protein, caspase-3 protein and caspase-6 mRNA expression fol-lowing amikacin treatment. （-）-Epigallocatechin-（3）-gallate （EGCG） inhibited SGN Bax protein, caspase-3 protein and caspase-6 mRNA expression, and enhanced Bcl-2 protein expression, thereby decreasing SGN apoptosis. Results demonstrated that EGCG can protect SGNs against amikacin-induced injury.

Effects of magnesium sulfate on neuron apoptosis and expression of caspase-3,bax and bcl-2 after cerebral ischemia-reperfusion injury

Objective To investigate the effects of magnesium sulfate on neuron apoptosis and the expressions of caspase-3,bax and bcl-2 after cerebral ischemia-reperfusion injury. Methods Thirty-six gerbils were randomly divided into three groups: Sham-operation group (So,n=12),ischemia-reperfusion group (I-R,n=12) and magnesium sulfate group (Ms,n=12). The neuron apoptosis was detected by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-fluorescence nick end labeling (TUNEL stain),and the protein expressions of caspase-3,bax and bcl-2 were detected by immunohistochemisty stain.Results Apoptotic neurons and the expressions of caspase-3 and bax were significantly increased in I-R and Ms groups,compared with those in So group. Apoptotic neurons and the expressions of bax and caspase-3 in Ms group were significantly less than those in I-R group. There was no significant difference in the expression of bcl-2 among three groups.Conclusions Magnesium sulfate decreases neuron apoptosis after cerebral ischemia-reperfusion injury,which may be related with the suppressed expressions of caspase-3 and bax.

MG132 Inhibits Myocardial Ischemia-reperfusion Injury by Regulating Apoptotic Pathway

Objectives To administrated proteasome inhibitor-MG-132 prior to reperfusion in rat myocardial ischemia-reperfusion model to determine whether MG-132 could reduce myocytic apoptosis. Methods and results MG-132 （0. 75 mg/kg in 2 ml DMSO） injection 5 min prior to reperfusion resulted significant reduction of myocardial reperfusion injury. This effect was accompanied by reduced polymorphonuclear neutrophils （PMN） infiltration in myocardial region surrounding the myocardial infarct, reduced apoptosis in cardiac myocytes, reduced NF-κB activation, as determined by electron microscopy, histology, immunohistochemistry, the terminal deoxynucleotidyl transferase-mediated nick endlabeling （TUNEL） method, reverse transcription-polymerase chain reaction. Functional effects of MG-132 on PMN accumulation, activation of nuclear factor kappa B （p65 mRNA and protein levels ）, and apoptosis were characterized in rat myocardial tissue. MG132 time-dependently inhibited myocardial p65 mRNA expression and reduced myocardial apoptotic index （AI） after reperfusion for 2 h, 6 h and 24 h （ P 〈 0. 01 ）. Moreover, MG-132 time-dependently decreased Bax protein levels, while increased Bcl-2 protein levels in ischemic and reperfused myocardium （ P 〈 0. 05 ）, its effect peaked after reperfusion for 24 h. Conclusions Our results demonstrate that MG-132 reduced myocardial reperfusion injury by inhibiting myosytic apoptotic cell death and blocking activation of NF-κB, down-regulating Bax expression and up-regulating Bcl-2 expression as well as el evating Bcl-2/Bax ratio.

Effect and mechanism of electronic magnetic pulse on peripheral lymphocytes in dogs

Objective To study the effects of electronic magnetic pulse (EMP) on peripheral lymphocytes in dogs and to explore the mechanisms of the biological effects of EMP.Methods T, TH and Ts lymphocytes were estimated by acid phosphatase cytochemistry. Apoptotic lymphocytes and Bax and Bcl-2 proteins related to apoptosis were observed with in situ terminal labeling and immunocytochemistry.Results Peripheral T lymphocyte subpopulations decreased obviously after EMP irradiation with (2 - 12) × 104 V/m. Apoptotic percentages of lymphocytes increased with the elevation of EMP doses. Ten days after different intensity radiation, the Bax protein was found to be elevated in accord with the peak value of lymphocyte apoptosis. However, Bcl-2 protein decreased obviously.Conclusion A definite field intensity EMP could induce injury to lymphocytes. Apoptosis induced by EMP is one of the main causes of peripheral lymphocyte death and leads to immunosuppression of the body.These results suggest that people should pay more attention to the injury caused by EMP, especially to the immunological functions of the body.